Due to its antimicrobial and healing-promoting effects, the application of cold atmospheric plasma (CAP) appears to be a promising modality in various fields of general medicine and dentistry. The aim of the present study was to evaluate the antibacterial and anti-biofilm activity of a handheld device utilizing ambient air for plasma generation. Suspensions of 11 oral bacteria (among them Fusobacterium nucleatum, Porphyromonas gingivalis, Parvimonas micra, Streptococcus gordonii, and Tannerella forsythia) were exposed to CAP for 10, 30, 60, and 120 s. Before and after treatment, colony forming unit (CFU) counts were determined. Then, 12-species biofilms were cultured on dentin and titanium specimens, and CAP was applied for 30, 60, and 120 s before quantifying CFU counts, biofilm mass, and metabolic activity. A reduction of ≥3 log10 CFU, was found for ten out of the eleven tested species at 30 s (except for T. forsythia) and for all species at 60 s. For biofilm grown on dentin and titanium specimens, the log10 reductions were 2.43 log10 CFU/specimen and by about 4 log10 CFU/specimen after 120 s of CAP. The CAP application did not reduce the biomass significantly, the metabolic activity of the biofilms on dentin and titanium decreased by 98% and 95% after 120 s of CAP. An application of 120 s of CAP had no cytotoxic effect on gingival fibroblasts and significantly increased the adhesion of gingival fibroblasts to the titanium surface. These results are promising and underline the potential of CAP for implementation in periodontal and peri-implantitis therapy.